Neurons extend neurites to communicate with other neurons or with their target tissues. This neuronal network in the adult central nervous system (CNS) regenerates only poorly after injury. This is a problem in the art, leading to poor patient outcomes following injury to the neuronal network.
Failure of the adult mammalian CNS to regenerate is due partly to the neurite outgrowth inhibitors associated with damaged myelin. Myelin-associated glycoprotein (MAG), Nogo-A (also known as Reticulon 4A) and oligodendrocyte myelin glycoprotein (OMgp) are myelin-associated inhibitors of neurite outgrowth that can bind to Nogo receptor 1 (NgR1). These myelin-associated proteins, Nogo-A, MAG and OMgp, transmit signals from oligodendrocytes into neurons through binding Nogo receptors. This Nogo signalling has critical roles in development and maintenance of the central nervous system (CNS). It can inhibit differentiation, migration, and neurite outgrowth of neurons, causing poor recovery of the adult CNS from damage.
Nogo-A binds to NgR1 through a domain called Nogo-66. The Nogo-66 domain is composed of 66 amino acids and the Nogo-66 domain alone, without other regions of Nogo-A, is sufficient to inhibit neurite outgrowth. MAG, but neither Nogo-A nor OMgp, can inhibit neurite outgrowth not only through NgR1 but also through NgR2, an NgR1 homologous protein (6).
NgR1 makes signalling complexes containing LINGO-1 and either p75NTR or TAJ/TROY (McGee, and Strittmatter Trends Neurosci 26, 193-198 (2003); Schwab et al. Trends Mol Med 12, 293-297 (2006)). Both p75NTR and TAJ/TROY belong to the TNFalpha receptor family and they are proposed to be the major components initiating intracellular signals for inhibition of neurite outgrowth. It is uncertain whether NgR2 makes complexes containing LINGO-1 and either p75NTR or TAJ/TROY.
While information on the roles of Nogo signalling is expanding, information on the mechanisms controlling this signalling is limited. This is a problem in the art. Increased intracellular levels of cAMP are known to overcome the inhibitory effects of Nogo signalling on neurite outgrowth (16). However, the detailed mechanism by which cAMP overcomes the effects of Nogo signalling is unknown.
BDNF, a member of the neurotrophin nerve growth factor family, not only stimulates neurite outgrowth of several types of neural cells in vitro (17-19, 20), but also partially promotes the recovery from spinal cord injury (21-24). Pre-treatment with BDNF increases the levels of intracellular cAMP in cultured neurons, allowing neurons to extend neurites even in the presence of the myelin-associated inhibitors (25). Furthermore, BDNF is implicated in injury-induced neurite sprouting in the hippocampus (26). These reports suggest that BDNF could potentially help regeneration of neuronal networks in the CNS even in the presence of the myelin-associated inhibitors of neurite outgrowth. However, the effect is limited and is not enough for complete regeneration of the neural networks.
A human neuroblastoma cell line SH-SY5Y shows BDNF-dependent neurite outgrowth after 5 days treatment with retinoic acid (RA) (18). SH-SY5Y cells initiate differentiation into neuron-like cells and start expression of neuron-specific proteins in response to RA. However, the neural cells differentiated from SH-SY5Y cells by RA show only limited morphological changes. BDNF treatment is required for efficient neurite outgrowth of the SH-SY5Y-derived neural cells, otherwise longer treatment with RA is required (27). Caesin kinase II (CK2) has been studied in the context of neurones. Caesin kinase II has been implicated in the phosphorylation of two different surface proteins in neurones. Neither protein is related to Nogo receptors. Furthermore, the studies in this area have been entirely dependent on the use of inhibitors of caesin kinase II. Thus, the function of intra-cellular CK2 has been studied in the art. Intra-cellular CK2 activity is known to be required for neurite outgrowth itself. Extra-cellular CK2 is known to exist. However, information on extra-cellular CK2 is largely unknown as noted above. In more detail, there are indications that amyloid beta precursor protein and neuroglican C can be phosphorylated at the surfaces of neurones by endogenous extra-cellular CK2. However, the effects of these phosphorylation(s) on neurite outgrowth (if any) are unknown.
Caesin kinase II has been used to treat collagen/laminin in certain in vitro preparations. These treatments have never involved cells. These treatments have only ever involved in vitro preparations of matrix proteins such as collagen or laminin.
No caesin kinase II treatment of cells is known in the prior art. Application of exogenous caesin kinase II to cells is not known in the prior art.
Ulloa et al (1993 EMBO vol 12 pp 1633-1640) inhibited CK2 activity in N2A mouse neuroblastoma cell line with antisense oligos and with a specific inhibitor. N2A cells extend neurites with neither retinoic acid (RA) nor BDNF. Using an N2A cell line, they found that neurite outgrowth from N2A cells is inhibited by depletion of CK2, and that phosphorylation of a microtuble-associated protein, MAP1B, is changed by the depletion. MAP is required for rearrangement of cytoskeleton, which is required for neurite outgrowth. Thus, they concluded that the change of MAP1B phosphorylation causes inhibition of neurite outgrowth by CK2 depletion. MAP1B phosphorylation is intra-cellular. Other proteins associated with rearrangement of cytoskeleton have been known to be phosphorylated by CK2, intra-cellularly. These intra-cellular phosphorylation events are required for neurite outgrowth itself. Since normal neurones, as well as N2A cells, can extend neurites without any stimulation, the inference is that these intra-cellular phosphorylation events are catalysed by a basal level of intro-cellular CK2 in neurones. The paper is not related to Nogo signaling.
No relationship between phosphorylation and Nogo signaling is known to date. The mechanism controlling Nogo signalling is unknown to date.
The present invention seeks to overcome problem(s) associated with the prior art.